The present invention relates to a novel protein having nitrile hydratase activity, the gene encoding the same, and a method for producing an amide from a nitrile via a transformant containing the gene. More specifically, the present invention relates to a novel protein having the activity of generating from a nitrile the corresponding amide; the gene encoding the same; a transformant transformed with an expression vector having a promoter sequence required at least for the expression of the protein; the DNA sequence encoding the protein and a terminator sequence; a method for expressing the protein via the culture of the transformant; a method for producing an amide by treating a nitrile with a culture broth containing the transformant or protein itself; an isolated microorganism; a treated microorganism; and protein or the immobilized products thereof.
For the technique to convert the nitrile groups of nitriles into amide groups by hydration, thereby producing the corresponding amides, there have been known a heating process using an acid or an alkali as a reaction catalyst and a heating process using copper catalysts. In recent years, however, a method has been adopted using a bacterial catalyst containing a water-soluble enzyme protein, namely nitrile hydratase, catalyzing the hydration reaction, (see Japanese Patent Applications Laid-open Publication Nos. 2693/1984 and 86889/1989; European Patent Applications Publication Nos. 188,316, 204,555, 444,639, and 93,782; U.S. Pat. Nos. 5,130,235 and 4,001,081; U.K. Patent Applications Publication Nos. 2,018,240, 2,076,820, and 2,076,821). It is believed that the method using the bacterial catalyst is superior to other methods, in that the hydration reaction can be carried out at ambient temperature and a higher conversion ratio can be achieved according to the method.
In using a bacterial catalyst, however, numerous considerations are required in preparing the bacterial catalyst, depending on the biological features of the bacterial catalyst to be used, such as the safety assessment of the bacterium, the optimum culture condition, and the like. Therefore, the activity as a biological catalyst is not necessarily readily increased to a level industrially applicable from the respect of cost.
As a method for improving such bacterial catalyst processes, a process is under investigation, comprising preparing a bacterial catalyst, namely an enzyme nitrile hydratase per se, via genetic engineering and converting nitriles into the corresponding amides. For example, Japanese Patent Application Laid-open No. 137688/1988 describes at an enzyme level a type of nitrile hydratase composed of two heterogeneous subunits and derived from genus Rhodococcus; Japanese Patent Application Laid-open No. 119778/1990 discloses a nitrile hydratase gene composed of two heterogeneous subunits and derived from genus Rhodococcus; and European Patent Application Publication No. 444,639 discloses a nitrile hydratase gene composed of two heterogeneous subunits and derived from genus Pseudomonas; and European Patent application Publication No. 455,646 discloses a nitrile hydratase gene composed of two heterogeneous subunits and derived from Rhodococcus rhodochrous.
For the purpose of preparing a bacterial catalyst with a higher catalytic activity, the present inventors have made investigations so as to produce a large quantity of a protein having an extremely high hydration activity of nitriles, namely nitrile hydratase activity, in a transformant via recombinant DNA technology. Consequently, the inventors have isolated and purified for the first time a novel protein useful for such an objective (often referred to as "nitrile hydratase protein", "nitrile hydratase AM24 protein", or "AM 24 protein" hereinbelow). Furthermore, the present inventors have separated and isolated the gene encoding the protein. Additionally, they have integrated the gene into an expression vector to generate a transformant, and have successfully achieved the large scale production of the nitrile hydratase protein via the transformant. Thus, the inventors have accomplished the present invention.